2018 the endonucleolytic rna cleavage function of nsp1 of middle east respiratory syndrome...
Post on 11-Sep-2021
3 Views
Preview:
TRANSCRIPT
1
1
The endonucleolytic RNA cleavage function of nsp1 of Middle East respiratory syndrome 2
coronavirus promotes the production of infectious virus particles in specific human cell 3
lines 4
5
Keisuke Nakagawa1, Krishna Narayanan
1, Masami Wada
1, Vsevolod L. Popov
2, 6
Maria Cajimat2, Ralph S. Baric
7 and Shinji Makino
1,3,4,5,6,# 7
Department of Microbiology and Immunology1, Department of Pathology
2, Center for 8
Biodefense and Emerging Infectious Diseases3, UTMB Center for Tropical Diseases
4, Sealy 9
Center for Vaccine Development5, and The Institute for Human Infections and Immunity
6, The 10
University of Texas Medical Branch, Galveston, Texas, USA, and Department of Epidemiology, 11
Department of Microbiology and Immunology, School of Medicine, University of North 12
Carolina at Chapel Hill7 13
Running title: MERS-CoV nsp1 affects virus assembly efficiency 14
15
#: Corresponding author: Shinji Makino 16
Corresponding author’s Mailing Address: 4.142E Medical Research Building 301 University 17
Boulevard, Galveston, Texas 77555-1019 18
Tel/Fax: (409) 772-2323/(409) 772-5065 19
E-mail: shmakino@utmb.edu 20
21
22
23
JVI Accepted Manuscript Posted Online 15 August 2018J. Virol. doi:10.1128/JVI.01157-18Copyright © 2018 American Society for Microbiology. All Rights Reserved.
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
2
Abstract 24
Middle East respiratory syndrome coronavirus (MERS-CoV) nsp1 suppresses host gene 25
expression in expressed cells by inhibiting translation and inducing endonucleolytic cleavage of 26
host mRNAs, the latter of which leads to mRNA decay. We examined the biological functions of 27
nsp1 in infected cells and its role in virus replication by using wild-type (wt) MERS-CoV and 28
two mutant viruses having specific mutations in the nsp1; one mutant lacked both biological 29
functions, while the other lacked the RNA cleavage function but retained the translation 30
inhibition function. In Vero cells, all three viruses replicated efficiently with similar replication 31
kinetics, while wt virus induced stronger host translational suppression and host mRNA 32
degradation than the mutants, demonstrating that nsp1 suppressed host gene expression in 33
infected cells. The mutant viruses replicated less efficiently than wt virus in Huh-7 cells, HeLa-34
derived cells, and 293-derived cells, the latter two of which stably expressed a viral receptor 35
protein. In 293-derived cells, the three viruses accumulated similar levels of nsp1 and major viral 36
structural proteins and did not induce IFN-β and IFN-λ mRNAs, however, both mutants were 37
unable to generate intracellular virus particles as efficiently as wt virus, leading to inefficient 38
production of infectious viruses. These data strongly suggest that the endonucleolytic RNA 39
cleavage function of the nsp1 promoted MERS-CoV assembly and/or budding in a 293-derived 40
cell line. MERS-CoV nsp1 represents the first CoV gene 1 protein that plays an important role in 41
virus assembly/budding and is the first identified viral protein whose RNA cleavage-inducing 42
function promotes virus assembly/budding. 43
44
45
46
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
3
Importance 47
MERS-CoV represents a high public health threat. Because CoV nsp1 is a major viral virulence 48
factor, uncovering the biological functions of MERS-CoV nsp1 could contribute to our 49
understanding of MERS-CoV pathogenicity and spur development of medical countermeasures. 50
Expressed MERS-CoV nsp1 suppresses host gene expression, but its biological functions for 51
virus replication and effects on host gene expression in infected cells are largely unexplored. We 52
found that nsp1 suppressed host gene expression in infected cells. Our data further demonstrated 53
that nsp1, which was not detected in virus particles, promoted virus assembly or budding in a 54
293-derived cell line, leading to efficient virus replication. These data suggest that nsp1 plays an 55
important role in MERS-CoV replication and possibly affects virus-induced diseases by 56
promoting virus particle production in infected hosts. Our data, which uncovered an unexpected 57
novel biological function of nsp1 in virus replication, contribute to further understanding of the 58
MERS-CoV replication strategies. 59
60
61
62
63
64
65
66
67
68
69
70
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
4
Introduction 71
Middle East respiratory syndrome (MERS) is a viral respiratory illness caused by MERS 72
coronavirus (MERS-CoV), which was first identified in Saudi Arabia in 2012 (1). MERS 73
outbreaks continue with increasing geographical distribution (2), and the mortality rate of MERS 74
is approximately 36% (http://www.who.int/emergencies/mers-cov/en/). MERS-CoV represents a 75
high public health threat, yet no vaccine or specific treatment for MERS is currently available. 76
CoVs belong to the order Nidovirales in the family Coronaviridae, and are currently 77
classified into four genera, alpha, beta, gamma, and delta CoVs. CoV is an enveloped virus 78
carrying a large single-stranded, non-segmented RNA with the 5’-end capped and the 3’-end 79
polyadenylated (3-5). Replication of MERS-CoV, a beta CoV, starts with binding of the virus 80
particle to a receptor, dipeptidyl peptidase 4 (6), which is also called CD26. After virus-host 81
membrane fusion (7), the viral genomic RNA is released into the cytoplasm and undergoes 82
translation of partially overlapping two large precursor polyproteins from gene 1, which 83
encompasses the 5’ two thirds of the genome. These precursor polyproteins are proteolytically 84
processed by two virally encoded proteinases to generate 16 mature proteins, non-structural 85
protein (nsp) 1 to 16 (8). All of these gene 1 proteins, except for nsp1 (9) and nsp2 (10), are 86
considered to be essential for CoV RNA synthesis (11). MERS-CoV replication results in 87
accumulation of eight viral mRNAs, including mRNA 1, the intracellular forms of viral genome, 88
and subgenomic mRNAs 2-8 (12, 13); these viral mRNAs form the 3’ co-terminal nested 89
structure and all carry the same leader sequence of ~70 nucleotides at the 5’-end (14-16). Viral 90
structural proteins (S, E, M, and N proteins) and four accessory proteins (3, 4a, 4b, and 5 91
proteins) are translated from these subgenomic mRNAs. MERS-CoV accessory proteins are not 92
essential for virus replication, yet they affect viral pathogenicity (17-19). Accumulation of viral 93
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
5
proteins and mRNA 1 leads to the assembly of virus particles and budding of virus particles at 94
endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes (20-22), followed 95
by subsequent release of the virus from the cells. CoV M protein plays a central role in virus 96
assembly (23-29). In many CoVs, including MERS-CoV, E protein, a low abundant protein in 97
the virus particle, is essential for production of infectious virus particles (23, 30-32), while 98
severe acute respiratory syndrome CoV (SARS-CoV) mutant lacking E protein is viable but 99
attenuated in growth (33). 100
Among the four CoV genera, only alpha and beta CoVs encode nsp1 (34). In contrast to 101
nsps 3-16 that play essential roles in exert viral RNA synthesis, nsp1 shares low amino acid 102
homology among CoVs (35-39) and the sizes of beta CoV nsp1 and alpha CoV nsp1 differ; the 103
former and the latter were ~28 kDa and ~9 kDa, respectively. Nonetheless, structural analysis 104
suggests that CoV nsp1 has a common origin (36) and nsp1 of alpha and beta CoVs share a 105
biological function to inhibit host gene expression. Past studies suggest that mechanisms of host 106
gene expression suppression induced by nsp1 of each CoV species may differ (39-43). Among 107
CoV nsp1s, mechanisms of nsp1-induced host gene suppression have been well characterized in 108
severe respiratory syndrome CoV (SARS-CoV) nsp1. SARS-CoV nsp1 is a cytoplasmic protein 109
that binds to the 40S ribosomal subunit (40, 41) and inactivates its translation function, which 110
leads to translation inhibition. The SARS-CoV nsp1-40S ribosome complex also induces 111
endonucleolytic cleavage of host mRNAs. Host 5’-3’ exonuclease, Xrn 1, further degrades host 112
mRNAs that undergo the nsp1-induced RNA cleavage (44). Although nsp1 suppresses 113
translation of SARS-CoV mRNAs, it does not induce endonucleolytic cleavage of SARS-CoV 114
mRNAs (45). Like SARS-CoV nsp1, expressed MERS-CoV nsp1 suppresses translation and 115
induces endonucleolytic RNA cleavage of host mRNA, leading to host mRNA decay (43). 116
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
6
However, unlike SARS-CoV nsp1, MERS-CoV nsp1 localized in both the cytoplasm and 117
nucleus, does not bind to 40S ribosomes, and targets host mRNAs of the nuclear origin, but not 118
mRNAs of cytoplasmic origin (43). Several lines of evidence point towards the strong possibility 119
that nsp1 is a major virulence factor of CoVs. SARS-CoV nsp1 suppresses the host innate 120
immune functions by inhibiting interferon (IFN) expression (46) and host antiviral signaling 121
pathways in infected cells (47). Nsp1 of porcine epidemic diarrhea virus (PEDV) suppresses type 122
II IFN (48). The contribution of nsp1 in CoV pathogenesis has been demonstrated for mouse 123
hepatitis virus (MHV) and SARS-CoV (49-51). 124
Although it has been considered that nsp1 is not essential for CoV RNA synthesis (9), the 125
biological roles of nsp1 in CoV replication are not well understood (39-41, 46, 52, 53). Our 126
present study demonstrated that, like in expressed cells, MERS-CoV nsp1 suppressed host gene 127
expression in infected cells. Unexpectedly, our studies revealed that the RNA cleavage function 128
of the MERS-CoV nsp1 promoted virus assembly or budding in a 293-derived cell line. To our 129
knowledge, MERS-CoV nsp1 is the first recognized CoV gene 1 protein that plays an important 130
role in the production of infectious virus particles. Furthermore, MERS-CoV nsp1 is the first 131
viral protein whose RNA cleavage-inducing function promoted the assembly/budding of virus 132
particles. 133
134
Results 135
Generation of MERS-CoV nsp1 mutants lacking host gene suppression functions. Toward 136
understanding the roles of MERS-CoV nsp1 in host gene expression and virus replication, we 137
aimed to generate a MERS-CoV nsp1 mutant that lacks both host mRNA cleavage and host 138
mRNA translation inhibition functions. Because alanine substitutions of two charged amino acid 139
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
7
residues, K164 and H165, of 180 amino-acid long SARS-CoV nsp1 abolish translation inhibition 140
function and the endonucleolytic RNA cleavage function (46), we hypothesized that alanine 141
substitution of a charged amino acid residue(s) near the C-terminal region of MERS-CoV nsp1 142
(193 amino-acid long) would also disrupt the MERS-CoV nsp1’s host gene suppression 143
functions. As alignment of amino acid sequences of MERS-CoV nsp1 and SARS-CoV nsp1 144
showed that K181 of MERS-CoV nsp1 corresponded to K164 of SARS-CoV nsp1, we 145
hypothesized that K181A mutation in MERS-CoV nsp1 would disrupt the host gene suppression 146
functions and constructed a T7 plasmid that expressed transcripts encoding MERS-CoV nsp1 147
with K181A mutation (MERS-CoV nsp1-mt). 148
To investigate the biological functions of MERS-CoV nsp1-mt, we independently 149
transfected 293 cells with capped and polyadenylated RNA transcripts encoding 150
chloramphenicol acetyltransferase (CAT), SARS-CoV nsp1, wild type MERS-CoV nsp1 151
(MERS-CoV nsp1-WT), MERS-CoV nsp1-mt, and MERS-CoV nsp1 mutant carrying R125A 152
and K126A mutation (MERS-CoV nsp1-CD), the latter of which lacks the endonucleolytic RNA 153
cleavage activity, but retains the translation suppression function (43). All encoded proteins 154
carried a C-terminal myc-tag. The cells were radiolabeled with Tran35
S-label from 8.5 h to 9.5 h 155
after transfection and cell extracts were subjected to SDS-PAGE analysis. Consistent with our 156
previous reports (43, 46), expression of SARS-CoV nsp1, MERS-CoV nsp1-WT, and MERS-157
CoV nsp1-CD suppressed host protein synthesis (Fig. 1A, top two panels). In contrast, MERS-158
CoV nsp1-mt protein expression did not inhibit host protein synthesis. We also confirmed the 159
expression of CAT, SARS-CoV nsp1, MERS-CoV nsp1-WT, MERS-CoV nsp1-CD and MERS-160
CoV nsp1-mt (Fig. 1A, bottom two panels). 161
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
8
Next, we tested the effect of MERS-CoV nsp1-mt expression on abundance of a host 162
mRNA. First, 293 cells were transfected with the RNA transcripts as described above. 163
Intracellular RNAs were extracted at 9 h post-transfection and subjected to Northern blot 164
analysis using a probe detecting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA 165
(Fig. 1B). Reduction of GAPDH mRNA abundance occurred in cells expressing SARS-CoV 166
nsp1 or MERS-CoV nsp1-WT, but not in those expressing MERS-CoV nsp1-CD or CAT (43). 167
MERS-CoV nsp1-mt expression also did not induce reduction in the abundance of GAPDH 168
mRNA, suggesting that MERS-CoV-mt did not induce the endonucleolytic RNA cleavage to 169
GAPDH mRNA and subsequent mRNA degradation. 170
To establish that MERS-CoV nsp1-mt lacks the endonucleolytic RNA cleavage function, 171
293 cells were transfected with a plasmid encoding CAT, MERS-CoV nsp1-WT, MERS-CoV 172
nsp1-CD, or MERS-CoV nsp1-mt, together with a plasmid encoding a bicistronic reporter 173
mRNA (Ren-EMCV-FF RNA) carrying the encephalomyocarditis virus internal ribosomal entry 174
sites (EMCV IRES) between the upstream Renilla luciferase (rLuc) gene and the downstream 175
Firefly luciferase (fLuc) gene (Fig. 1C, top panel); all expressed proteins carried a C-terminal 176
myc tag. SARS-CoV nsp1 and MERS-CoV nsp1-WT served as positive controls as they induce 177
endonucleolytic RNA cleavage within the EMCV IRES region of Ren-EMCV-FF RNA (40, 43, 178
45), while CAT and MERS-CoV nsp1-CD served as negative controls. Intracellular RNAs were 179
extracted at 24 h post-transfection and subjected to Northern blot analysis using rLuc probe. 180
Expression of MERS-CoV nsp1-WT and SARS-CoV nsp1 induced endonucleolytic cleavage of 181
Ren-EMCV-FF RNA, generating a fast migrating RNA fragment (Fig. 1C, second panel; see 182
arrowhead) and reduction in the amounts of the full-length Ren-EMCV-FF RNA (Fig. 1C, 183
second panel; see arrow). Consistent with our previous report (43), SARS-CoV nsp1 was more 184
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
9
active than MERS-CoV nsp1-WT for inducing RNA cleavage. The RNA fragment was absent in 185
cells expressing the MERS-CoV nsp1-mt, demonstrating that the MERS-CoV nsp1-mt lacked 186
the endonucleolytic RNA cleavage activity. Western blat analysis confirmed expression of 187
SARS-CoV nsp1, MERS-CoV nsp1-WT, MERS-CoV nsp1-CD and MERS-CoV nsp1-mt in 188
transfected cells (Fig. 1C, fourth panel). Consistent with our previous report (43), SARS-CoV 189
nsp1 and MERS-CoV nsp1-WT accumulated poorly in expressed cells; probably these nsp1s 190
targeted their own template mRNAs for degradation, leading to poor protein accumulation. 191
MERS-CoV nsp1-CD, which is deficient for the endonucleolytic RNA cleavage function 192
(43), suppressed host translation (Fig. 1A), demonstrating that MERS-CoV nsp1-CD retained its 193
translational suppression function. Absence of host translation inhibition in cells expressing 194
MERS-CoV nsp1-mt demonstrated that MERS-CoV nsp1-mt lost both the RNA cleavage 195
function and the translation suppression function. 196
197
Replication of MERS-CoV mutants encoding mutant nsp1 in Vero cells. To explore the role 198
of nsp1 in virus replication and host gene expression, we rescued MERS-CoV-WT encoding 199
MERS-CoV nsp1-WT, MERS-CoV-CD carrying MERS-CoV nsp1-CD, and MERS-CoV-mt 200
carrying MERS-CoV nsp1-mt by using a reverse genetics system (54). All three viruses 201
replicated efficiently with similar replication kinetics in Vero cells (Fig. 2A). Also, all of the 202
viruses accumulated similar levels of viral structural proteins, S, M, and N, nsp1, and virus-203
specific mRNAs at each indicated time point (Fig. 2B and C). 204
Next, we examined the effects of nsp1 for host mRNA stability and host protein synthesis 205
in infected Vero cells. The abundance of host GAPDH mRNA was lower in MERS-CoV-WT-206
infected cells than in MERS-CoV-CD- and MERS-CoV-mt-infected cells (Fig. 3A). Replication 207
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
10
of MERS-CoV-WT, but not the two mutant viruses, in the presence of actinomycin D (ActD), 208
also resulted in reduced GAPDH mRNA levels (Fig. 3B, right panel), demonstrating that nsp1 209
induced efficient degradation of preexisting GAPDH mRNA in infected cells. Metabolic 210
radiolabeling experiments showed that replication of MERS-CoV-WT as well as the two mutant 211
viruses induced an inhibition of host protein synthesis. (Fig. 3C). Although the extent of host 212
translation inhibition induced by these viruses was modest at 24 h p.i., a stronger inhibition of 213
host translation was observed in MERS-CoV-WT-infected cells than in those infected with the 214
mutant viruses at 32 h p.i., suggesting that the strong inhibition of host gene expression was due 215
to a combined effect of the nsp1-mediated RNA cleavage and the translation suppression 216
function. Taken together, these data established that nsp1 suppressed host gene expression by 217
inducing host mRNA decay and inhibiting host translation in infected cells. 218
219
Replication of MERS-CoV-WT and the mutant viruses in various cell lines. We 220
subsequently examined replication kinetics of the three viruses in various cell lines. All of the 221
three viruses replicated efficiently with similar replication kinetics in Calu-3 cells, a human 222
airway epithelial cell line (55), regardless of multiplicity of infections (MOIs) (Fig. 4A). The 223
three viruses replicated efficiently and similarly at an MOI of 3 in Huh-7 cells, a well 224
differentiated hepatocyte derived cellular carcinoma cell line (56), except that the titer of MERS-225
CoV-WT was statistically ~10 fold higher at peak titers than those of the mutants at 32 h p.i. (Fig. 226
4B). In contrast, the two mutant viruses replicated ~2 logs less efficiently than MERS-CoV-WT 227
in Huh-7 cells at an MOI of 0.01 (Fig. 4B). The titers of MERS-CoV-WT were statistically 228
higher than those of the mutant viruses from 24 to 48 h p.i. at an MOI of 3 in 293 cells stably 229
expressing human CD26 (293/CD26 cells) (Fig. 4C). Likewise, both mutant viruses replicated 230
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
11
less efficiently than MERS-CoV-WT in 293/CD26 cells at an MOI of 0.01. MERS-CoV-WT 231
also replicated to statistically higher titers than the two mutants throughout the infection at an 232
MOI of 3 in HeLa cells stably expressing CD26 (HeLa/CD26 cells), while both mutants showed 233
similar titers and replication kinetics. At an MOI of 0.01 in HeLa/CD26 cells, MERS-CoV-WT 234
replicated to higher titers than the mutant viruses after 12 h p.i. Taken together, these data 235
suggested that nsp1 promoted virus replication in a cell type-dependent manner. 236
237
Replication of the three viruses does not induce IFN-β and IFN-λ mRNAs. To determine the 238
mechanisms of nsp1-mediated promotion of virus replication, we used 293/CD26 cells for 239
subsequent analyses. We first tested the possibility that the mutant nsp1 did not suppress the host 240
innate immune responses, thereby promoting production of type I IFN and/or III IFN and leading 241
to inhibition of virus replication. To this end, we examined induction of IFN-β and IFN-λ 242
mRNAs in infected 293/CD26 cells (Fig. 5). Sendai virus (SeV) infection resulted in efficient 243
induction of IFN-β and IFN-λ mRNAs, whereas mock-infected cells did not. None of the three 244
viruses efficiently induced IFN-β and IFN-λ mRNAs from 8 h to 32 h p.i., suggesting that 245
inefficient replication of MERS-CoV-mt and MERS-CoV-CD was not due to induction of type I 246
and III IFNs and that the host gene suppression functions of MERS-CoV nsp1 did not play a 247
significant role in inhibiting the induction of IFN-β and IFN-λ mRNAs in 293/CD26 cells. 248
249
Accumulation of viral proteins and mRNAs in infected 293/CD26 cells. To discern whether 250
inefficient replication of mutant viruses in 293/CD26 cells was due to poor accumulation of viral 251
structural proteins, we examined the abundance of major viral structural proteins, including S, M, 252
and N proteins. No substantial differences in the accumulation of these structural proteins were 253
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
12
noted among cells infected with the three viruses (Fig. 6A). The three viruses also accumulated 254
similar levels of nsp1 (Fig. 6A). 255
Northern blot analysis showed that accumulation of viral mRNAs were marginally higher 256
in MERS-CoV-WT-infected cells than in cells infected with the mutant viruses (Fig. 6B). In 257
addition to eight mRNA species, we noted presence of two additional viral-specific RNA bands, 258
one migrated faster than mRNA 2 and the other migrated faster than mRNA 5, in MERS-CoV-259
mt-infected cells. We also detected another viral-specific RNA band that migrated between 260
mRNA 5 and mRNAs 6/7 in MERS-CoV-WT-infected cells at 24 h p.i. The origins of these viral 261
RNAs are currently unclear, yet they may represent defective RNAs or subgenomic mRNAs. 262
qRT-PCR analyses revealed that mRNA 1 of MERS-CoV-WT accumulated higher abundance 263
than that of MERS-CoV-mt at 8 h, 16 h, and 32 h p.i. and that of MERS-CoV-CD at 32 h p.i. 264
Additionally, the amount of mRNA 8 of MERS-CoV-WT was higher than those of the mutant 265
viruses at 8 and 32 h p.i. (Fig. 6C). These studies showed that there was a trend that MERS-CoV-266
WT accumulated higher levels of viral mRNAs than the mutant viruses. 267
268
Analyses of host gene expression in infected 293/CD26 cells. The effects of nsp1 for host 269
mRNA stability and host protein synthesis in infected 293/CD26 cells were examined next. 270
Replication of both mutant viruses did not affect abundance of GAPDH mRNA, while the 271
abundance of GAPDH mRNA was substantially reduced in MERS-CoV-WT-infected cells (Fig. 272
7A), suggesting that MERS-CoV nsp1, but not MERS-CoV nsp1-CD and MERS-CoV nsp1-mt, 273
induced degradation of GADPH mRNA in infected cells. Metabolic radiolabeling experiments 274
showed that replication of all three viruses induced host protein synthesis inhibition at 24 h p.i. 275
(Fig. 7B). Because MERS-CoV nsp1-mt were deficient for the translation inhibition and mRNA 276
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
13
cleavage functions (Fig. 1), host translational suppression in MERS-CoV-mt-infected cells was 277
independent from the nsp1 function. MERS-CoV-WT and MERS-CoV-CD induced slightly 278
stronger host translational suppression than MERS-CoV-mt at 24 h p.i., suggesting that the 279
translation suppression function of the nsp1 modestly contributed to host translation suppression 280
in 293/CD26 cells. 281
282
Titers of cell-associated virus and abundances of released virus particles among the three 283
viruses. MERS-CoV-WT replicated to higher titers than two mutant viruses in 293/CD26 cells 284
(Fig. 4C), whereas accumulation of intracellular viral structural proteins were comparable among 285
the three viruses (Fig. 6A). One possible interpretation of these data was that the mutant viruses 286
were able to undergo assembly and budding of infectious virus particles as efficient as MERS-287
CoV-WT, yet the mutant viruses were unable to efficiently release infectious viruses from 288
infected cells. If this is the case, the titers of the cell-associated viruses would be similar among 289
the three viruses. We found that the titers of cell-associated MERS-CoV-WT were higher than 290
those of the cell-associated mutant viruses at 24, 36, and 48 h p.i (Fig. 8A). Also, the titers of 291
cell-associated MERS-CoV-CD were higher than those of cell-associated MERS-CoV-mt at 36 292
and 48 h p.i. (Fig. 8A). Low titers of cell-free and the cell-associated viruses (Fig. 4C and 8A) in 293
mutant viruses suggested that the release of infectious viruses was not selectively inhibited in the 294
mutant viruses. Rather, these data implied that low titers of cell-free viruses in the mutant virus-295
infected cells was due to accumulation of low titers of cell-associated viruses. 296
Because soluble CD26 binds to MERS-CoV particles and neutralizes virus infectivity 297
(57), expressed CD26 might have bound to intracellular virus particles in the mutant virus-298
infected cells, leading to neutralization of the cell-associated virus particles and/or preventing 299
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
14
virus release. In contrast, MERS-CoV nsp1-WT expression might have efficiently suppressed 300
CD26 expression in MERS-CoV-WT-infected cells, preventing the putative binding of CD26 to 301
intracellular virus particles. To test a likelihood of this possibility, we examined abundance of 302
CD26 in infected cells (Fig. 8B). While replication of the three viruses did not affect the levels of 303
CD26 at 24 h p.i., it caused reduction of CD26 abundance at 36 h p.i. There were no substantial 304
differences in the amounts of CD26 among MERS-CoV-WT-infected cells and mutant virus-305
infected cells at both time points. These data showed that CD26 expression levels did not play 306
significant roles in low titers of cell-associated and cell-free viruses in the mutant viruses. 307
Although infection with the mutant viruses produced low titers of infectious viruses, it is 308
possible that high levels of noninfectious viruses could have been released into the supernatant 309
from the cells infected with the mutant viruses. To test this possibility, we examined the amount 310
of virus particles, including both infectious and noninfectious, that are released from infected 311
cells. We harvested culture fluid from infected 293/CD26 cells, inactivated the released viruses 312
by 60
Co irradiation, purified the virus particles by sucrose gradient centrifugation, and estimated 313
the amounts of the released viruses by Western blot analysis using antibodies detecting S, M, and 314
N proteins. Substantially stronger signals of S, M, and N proteins were observed in the purified 315
virus particles obtained from MERS-CoV-WT-infected cells showed than in those obtained from 316
mutant virus-infected cells (Fig. 8C), suggesting that the amount of virus particles, including 317
both infectious and noninfectious, released from the mutant virus-infected cells were lower than 318
those released from MERS-CoV-WT-infected cells. As virus inactivation by gamma irradiation 319
is believed to be mainly caused by radiolytic cleavage or crosslinking of genetic material (58-62), 320
we did not examine the amount of viral genomic RNA in the purified 60
Co-irradiated virus 321
particles. 322
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
15
323
Transmission electron microscopic analysis of infected 293/CD26 cells. To further 324
understand the mechanism of inefficient replication of mutant viruses, we performed 325
transmission electron microscopic analysis of infected 293/CD26 cells (Fig. 9). We observed the 326
accumulation of intracellular virus particles, with the expected average size, within intracellular 327
vesicles (Fig. 9A-9C). We also noted the presence of particles, whose sizes were similar to virus 328
particles, outside of these vesicles, yet the identity of these particles was unclear. Because CoV 329
undergoes assembly and budding of virus particles at ERGIC membranes (20-22) and then 330
follows the secretory pathway for egress (63), these vesicles containing virus particles most 331
probably represented those in the secretory pathway. Counting the number of intracellular virus 332
particles in an arbitrarily selected 30 vesicles for each virus showed the presence of statistically 333
lower numbers of virus particles within these vesicles inside the cells infected with the mutant 334
viruses versus MERS-CoV-WT-infected cells (Fig. 9D). However, the number of virus particles 335
within these virus-containing vesicles between MERS-CoV-CD-infected cells and MERS-CoV-336
mt-infected cells showed no statistical difference. These data suggested that the mutant viruses 337
were less efficient at production of virus particles, including virus assembly and/or virus budding, 338
than MERS-CoV-WT. Taken together, these data support a notion that the mutant viruses were 339
able to accumulate viral structural proteins as efficiently as MERS-CoV-WT in 293/CD26 cells 340
(Fig. 6A), whereas they were inefficient for assembly or budding of virus particles (Fig. 9). This 341
resulted in low titers of cell-associated viruses (Fig. 8A) and release of a low number of virus 342
particles (Fig. 8C), including infectious viruses (Fig. 4C). 343
As our studies revealed the importance of nsp1 for production of virus particles, we also 344
explored the possibility that MERS-CoV nsp1 promotes virus assembly/budding by 345
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
16
incorporating itself into virus particles. Western blot analysis of purified MERS-CoV-WT, 346
MERS-CoV-CD, and MERS-CoV-mt using anti-nsp1 antibody did not show the presence of 347
nsp1 in the purified virus particles (data not shown), suggesting that MERS-CoV nsp1 was not 348
associated with virus particles or was not a major viral protein in the virus particles. 349
350
Discussion 351
The present study explored the biological significance of MERS-CoV nsp1 in virus 352
replication. By characterizing MERS-CoV nsp1-WT and MERS-CoV nsp-1-CD, the latter of 353
which lacked the endonucleolytic RNA cleavage function, our previous study showed that 354
expressed MERS-CoV nsp1 suppresses host gene expression by inducing endonucleolytic 355
cleavage of host mRNAs and inhibiting translation, the latter of which is independent from the 356
former function (43). SARS-CoV nsp1 also suppresses host gene expression by inducing 357
endonucleolytic cleavage of mRNAs and inhibiting translation, yet existing data imply that 358
MERS-CoV nsp1 and SARS-CoV nsp1 exert these functions by different mechanisms (43). 359
Namely, SARS-CoV nsp1, a cytoplasmic protein (41), binds to 40S ribosomal subunits, 360
inactivates translational function of the 40S ribosomes (40), and induces degradation of host 361
nucleus-derived mRNAs and cytoplasmically synthesized mRNAs (43). In contrast, MERS-CoV 362
nsp1 localizes in both the cytoplasm and nucleus (43), does not bind to 40S ribosomes and 363
induces degradation of mRNAs of nuclear origin, but not those of cytoplasmic origin (43). The 364
present study revealed that MERS-CoV nsp1-mt with K181A mutation lost the RNA cleavage 365
and translation inhibition functions (Fig.1). The K181A mutation corresponded to one of the 366
K164A and H165A mutations introduced in SARS-CoV nsp1-mt, which also lacks both RNA 367
cleavage and translation inhibition functions (46). These data suggest importance of the C-368
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
17
terminal regions of SARS-CoV nsp1 and MERS-CoV nsp1 for the biological functions. Because 369
SARS-CoV nsp1-mt is deficient for binding to 40S ribosomes (40), SARS-CoV nsp1 probably 370
interacts with the 40S ribosome through its C-terminal region. MERS-CoV nsp1’s selective 371
biological effects toward nucleus-derived mRNAs led us to hypothesize that MERS-CoV nsp1 372
targets nucleus-derived mRNAs, by binding to one of the mRNA-binding proteins that form the 373
host mRNP complex, and inhibits the expression of host genes (43). If this hypothesis is correct, 374
disruption of MERS-CoV nsp1’s functions by the K181A mutation imply that the MERS-CoV 375
nsp1 accesses the host mRNP complex through its C-terminal region. It is conceivable that both 376
MERS-CoV nsp1 and SARS-CoV nsp1 access target host protein/factors through their C-377
terminal regions. 378
Although MERS-CoV-WT, MERS-CoV-CD, and MERS-CoV-mt replicated efficiently 379
with similar growth kinetics in Vero and Calu-3 cells, the two mutant viruses replicated less 380
efficiently than MERS-CoV-WT in 293/CD26 cells, Huh-7 cells, and HeLa/CD26 cells (Fig. 4). 381
We explored whether induction of type I and/or III IFNs caused inefficient replication of the 382
mutant viruses in 293/CD26 cells, which were competent for induction of IFN- and IFN-λ 383
mRNAs by SeV infection (Fig. 5). Replication of the three viruses did not induce high levels of 384
IFN- and IFN-λ mRNAs (Fig. 5), demonstrating that inefficient replication of the two mutant 385
viruses was not due to induction of the type I and III IFNs. It has been reported that MERS-CoV 386
mutant lacking all accessory genes induced higher levels of IFN- IFN-λ1, and IFN-λ3 mRNAs 387
than wt MERS-CoV in Calu-3 2B4 cells (19). Accordingly, it is possible that other viral proteins, 388
including the accessory proteins, suppressed induction of IFN- and IFN-λ mRNAs in MERS-389
CoV-CD-infected 293/CD26 cells and MERS-CoV-mt infected 293/CD26 cells. In contrast to 390
MERS-CoV, SARS-CoV carrying biological inactive nsp1 induced high levels of IFN- mRNA 391
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
18
and type I IFN in infected cells (46). These data suggest that MERS-CoV and SARS-CoV use 392
different strategies to suppress induction of innate immune responses. 393
We observed that nsp1-induced changes in translational activities differed between infected 394
Vero cells (Fig. 3) and infected 293/CD26 cells (Fig. 7). For each cell line, levels of nsp1 395
accumulation were similar among the three viruses (Figs. 2B and 6A), suggesting that the 396
differences in the functions of nsp1, but not their expression levels, affected translational 397
activities. MERS-CoV-WT inhibited translation at 32 h p.i. in Vero cells, while the mutant 398
viruses induced modest and similar levels of translational inhibition (Fig. 3C), demonstrating that 399
MERS-CoV-nsp1, particularly its RNA cleavage function, played a significant role in 400
translational suppression in Vero cells. The moderate level of host translation inhibition in 401
MERS-CoV-mt-infected Vero cells (Fig. 3C) could be due to the induction of the cellular stress 402
response to virus infection, which is independent of the mode of translation inhibition induced by 403
nsp1. In contrast to Vero cells, nsp1 did not play a significant role in virus-induced translation 404
suppression in 293/CD26 cells, as the three viruses, including MERS-CoV-mt, efficiently 405
inhibited translation at 24 h p.i. (Fig. 7B). These data suggest a cell line-specific effect of MERS-406
CoV nsp1 on host gene expression. 407
There was a trend of increased viral mRNA accumulation in MERS-CoV-WT-infected 408
293/CD26 cells than in mutant virus-infected 293/CD26 cells (Figs. 6B, C), yet the differences in 409
the amounts of viral mRNAs did not determine the amount of viral proteins (Fig. 6A). As 410
translational activities in 293/CD26 cells infected with MERS-CoV-WT, -CD, or -mt were 411
similar and lower than that of mock-infected 293/CD26 cells at 24 h p.i. (Fig. 7B), low 412
translational activities might have served as a bottle neck, which allowed translation of only a 413
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
19
fraction of viral mRNAs in MERS-CoV-WT-infected 293/CD26 cells, resulting in similar 414
amounts of viral protein accumulation in the three viruses. 415
We explored the mechanism of inefficient replication of MERS-CoV-CD and MERS-CoV-416
mt in 293/CD26 cells. Low titers of cell-associated and cell-free viruses in mutant virus-infected 417
cells (Figs. 8 and 4C) were not due to inefficient accumulations of major viral structural proteins 418
and nsp1 (Fig. 6). The data showing similar levels of CD26 expression in 293/CD26 cells 419
infected with the three viruses (Fig. 8B) did not support a possibility that MERS-CoV-WT, but 420
not the mutant viruses, inhibited CD26 expression and prevented interaction of CD26 with 421
intracellular virus particles, which might have induced neutralization of intracellular virus 422
particles and/or inhibition of virus release. Electron microscopic analysis showed less efficient 423
intracellular virus particle accumulation in the virus-containing vesicles in mutant virus-infected 424
cells than in MERS-CoV-WT-infected cells (Fig. 9). These data strongly suggested that low 425
levels of virus particle accumulation, which was probably due to inefficient virus 426
assembly/budding, in mutant virus-infected cells caused low titers of cell-associated viruses (Fig. 427
8A) and cell-free viruses (Fig. 8B). The data that MERS-CoV-CD, expressing MERS-CoV nsp1-428
CD lacking the endonucleolytic RNA cleavage function and retaining the translation inhibition 429
function, was inefficient for accumulation of intracellular virus particles strongly suggested that 430
the RNA cleavage function of the nsp1 was required for efficient assembly/budding of MERS-431
CoV particles. To our knowledge, this is the first demonstration that a CoV gene 1 protein affects 432
efficiency of virus assembly. 433
Several different mechanisms are conceivable for inefficient assembly/budding of the two 434
mutant viruses in 293/CD26 cells. One possible mechanism may be that low accumulation of E 435
protein, which is known to be important for assembly of many CoVs (23, 30, 64), might have 436
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
20
occurred in mutant virus-infected cells and prevented efficient virus assembly. Absence of 437
appropriate anti-E protein antibodies prevented us from directly examining this possibility. 438
However, it seems illogical that the loss of the RNA cleavage function of the MERS-CoV nsp1, 439
which did not severely affect accumulation of S, M, and N proteins (Fig. 6A) and mRNA 6 440
encoding E protein (Fig. 6B), selectively suppressed E protein expression in mutant virus-441
infected 293/CD26 cells. Furthermore, similar translational activities in MERS-CoV-WT- and 442
mutant virus-infected 293/CD26 cells (Fig. 7B) did not support a possibility of selective 443
inhibition of E protein accumulation in the mutant virus-infected 293/CD26 cells. We suspect 444
that E protein accumulation was not low in mutant virus-infected 293/CD26 cells. 445
Another possible mechanism for inefficient assembly of the two mutant viruses could be 446
due to lower levels of mRNA 1 accumulation in the mutant virus-infected 293/CD26 cells (Figs. 447
6B and C). CoV-like particles are produced from cells expressing viral structural proteins in the 448
absence of mRNA 1 (23), yet it is unknown whether mRNA 1 affects efficiency of CoV particle 449
assembly. There was a trend of higher accumulation of mRNA 1 in MERS-CoV-WT-infected 450
293/CD26 cells than mutant virus-infected 293/CD26 cells (Fig. 6C). If mRNA 1 promotes 451
assembly of CoV particles, reduced amounts of mRNA 1 would have caused inefficient virus 452
assembly in mutant virus-infected 293/CD26 cells. Another possibility of the inefficient 453
assembly/budding of the two mutant viruses would be that MERS-CoV-nsp1-WT, but not 454
MERS-CoV-nsp1-CD and MERS-CoV-nsp1-mt, suppressed expression of a host protein that 455
restricts assembly/budding of MERS-CoV particles. Although host translation was inhibited to 456
similar levels between MERS-CoV-WT-infected 293/CD26 cells and MERS-CoV-CD-infected 457
293/CD26 cells (Fig. 7B), the RNA cleavage function of the MERS-CoV nsp1-WT might have 458
induced efficient degradation of the mRNA encoding this putative virus assembly/budding 459
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
21
restriction protein, preventing the accumulation of this putative protein and promoting virus 460
assembly/budding in MERS-CoV-WT-infected 293/CD26 cells. In contrast, due to lack of the 461
RNA cleavage function in MERS-CoV-nsp1-CD and MERS-CoV-nsp1-mt, this putative host 462
protein might have been expressed abundantly in mutant virus-infected 293/CD26 cells, 463
preventing efficient virus assembly. If this possibility is the case, a plausible reason for efficient 464
replication of mutant viruses in Vero and Calu-3 cells may be that these cells express the putative 465
virus restriction protein at low levels, amounts of which are not sufficient for inhibiting MERS-466
CoV assembly/budding. Tetherin has been known as a host restriction factor capable of impeding 467
the release of multiple viruses, including CoV (65-69). Because tetherin primarily prevents 468
release of viruses from the cells, but does not affect virus assembly/budding, this putative virus-469
assembly restriction protein may not be tetherin. 470
Viral proteins that inhibit host gene expression, including nsp1 of CoV (49-51), are often 471
major virulence factors (70-79). Accordingly, it is likely that MERS-CoV nsp1 also plays a 472
critical role in MERS-CoV pathogenesis. Because MERS-CoV nsp1-CD and MERS-CoV nsp1-473
mt negatively affected the efficient production of infectious viruses in several human cell lines, 474
MERS-CoV-CD and MERS-CoV-mt could exhibit a reduced virulence in infected hosts, 475
compared to MERS-CoV-WT, at least partly due to the inefficient production of infectious 476
viruses. Further studies are warranted, including a detailed characterization of the replication and 477
virulence of MERS-CoV-WT, MERS-CoV-CD and MERS-CoV-mt in animal models (80-83), to 478
clarify the role of nsp1 in MERS-CoV pathogenicity. 479
Herpesvirus simplex virus types 1 and 2 virion host shutoff protein, Kaposi’s sarcoma-480
associated herpesvirus SOX, and influenza A virus PA-X are virus-encoded RNases that induce 481
endonucleolytic cleavage of host mRNAs, leading to host mRNA degradation (84, 85). These 482
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
22
viral endonucleases contribute to evasion of host antiviral responses, including IFN response and 483
stress granule formation, and contribute to viral pathogenesis (70-79), while roles of these virus-484
encoded endonucleases in production of virus particle have not been explored. To our knowledge, 485
MERS-CoV nsp1 represents the first viral protein whose RNA cleavage-inducing function 486
promotes virus assembly/budding. 487
488
Materials and methods 489
Cells Vero cells (ATCC number CCL-81), Calu-3 cells, and Huh-7 cells were maintained in 490
minimum essential medium supplemented with 10% fetal calf serum (FBS), GlutaMAX (Gibco) 491
supplemented with 10% FBS, and Dulbecco modified Eagle medium supplemented with 10% 492
FBS, respectively. HeLa/CD26 cells were generated by following a previous report (43). Briefly, 493
HeLa cells were transfected with pCAGGS-CD26-BlasticidinR and grown in selection medium 494
containing blasticidin (10 µg/ml) for two weeks. 293/CD26 cells were generated by transfecting 495
pCAGGS-CD26-BlasticidinR into 293 cells (ATCC) and subsequent incubation of the 496
transfected cells in the presence of blasticidin (12 µg/ml). Stable expression of human CD26 in 497
293/CD26 and HeLa/CD26 cells were confirmed by Western blot analysis with anti-human 498
DPP4 antibody (R&D systems). 499
500
Viruses MERS-CoV-WT, MERS-CoV-CD, and MERS-CoV-mt were rescued by using reverse 501
genetics system as reported previously (54). All virus strains were passaged once in Vero cells 502
and used for infection studies. The presence of the expected mutation and absence of other 503
mutations in nsp1 was confirmed prior to use of these viruses. For virus growth analysis, Vero, 504
Calu-3, Huh-7, HeLa/CD26, and 293/CD26 cells were infected with MERS-CoV-WT, -CD or –505
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
23
mt at an MOI of 3 or 0.01. After virus adsorption for 1 h at 37°C, cells were washed twice with 506
PBS and incubated with the appropriate medium. The culture fluid was collected at indicated 507
time points, and the infectious virus titers were determined by plaque assay on Vero cells. All 508
experiments with infectious MERS-CoV were performed in an approved biosafety level 3 509
laboratory at The University of Texas Medical Branch at Galveston. Cantell strain of SeV was 510
obtained from Charles River Laboratory (Wilmington, MA), was used to infect cells at 100 511
hemagglutination (HA) unit/ml. Viral stocks were prepared in Vero cells and stored at -80°C. 512
513
Plasmids pCAGGA-based expression plasmids, pCAGGS-CAT, -SARS-CoV nsp1, -MERS-514
CoV nsp1-WT, and -MERS-CoV nsp1-CD, all of which carried a C-terminal myc tag, were 515
described previously (43). pCAGGS-MERS-CoV nsp1-mt, expressing a C-terminal myc-tagged 516
MERS-CoV nsp1 carrying a Lys-to-Ala substitution at position 181, was generated from 517
pCAGGS-MERS-CoV nsp1 by using a recombinant PCR-based method. Sequence analysis of 518
the plasmid confirmed the presence of the expected nsp1 sequence. pRL-EMCV-FL expressing a 519
bicistronic reporter mRNA carrying the EMCV IRES between the upstream rLuc gene and the 520
downstream fLuc gene was used (43). 521
522
Northern blot analysis Subconfluent 293 cells were transfected with a plasmid encoding CAT, 523
SARS-CoV nsp1, MERS-CoV nsp1-WT, -CD or –mt, together with pRL-EMCV-FL. At 24 h 524
post-transfection, total RNAs were extracted, and subjected to Northern blot analysis with an 525
rLuc probe and GAPDH probe. Northern blot analysis was performed as described previously 526
(43). Vero and 293/CD26 cells were infected with MERS-CoV-WT, MERS-CoV-CD, or MERS-527
CoV-mt at MOI of 3. At the indicated times p.i., total RNAs were extracted, and subjected to 528
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
24
Northern blot analysis with GAPDH probe. To detect MERS-CoV mRNAs, a DIG-labeled 529
random-primed probe corresponding to nt 29,084 to 29,608 of the MERS-CoV genome were 530
used. 531
532
Metabolic radiolabeling of intracellular proteins Subconfluent 293 cells were transfected 533
with in vitro-synthesized capped and polyadenylated RNA transcripts encoding CAT, SARS-534
CoV nsp1, MERS-CoV nsp1-WT, MERS-CoV nsp1-CD, or MERS-CoV nsp1-mt. All encoded 535
proteins carried the C-terminal myc tag. After incubation in methionine-deficient medium for 30 536
min, the cells were metabolically labeled with 20 μCi of Tran35
S-label (1,000 Ci/mmol; Perkin 537
Elmer)/ml from 8.5 to 9.5 h post-transfection. Infected Vero cells were radiolabeled with 75 μCi 538
of Tran35
S-label/ml for 1 h at 16 or 24 h p.i. The cell extracts were prepared by lysing the cells in 539
SDS-PAGE sample buffer. Cell lysates were subjected to SDS-PAGE analysis, followed by 540
autoradiography and colloidal Coomassie blue staining. 541
542
Western blot analysis Antibodies for MERS-CoV proteins were generated by immunizing 543
rabbits with the following synthetic peptides: NDITNTNLSRGRGRNPKPR for anti-MERS-544
CoV N protein peptide antibody, DDRTEVPQLVNANQYSPCVSIVC for anti-MERS-CoV S 545
protein peptide antibody, and CDYDRLPNEVTVAK for anti-MERS-CoV M protein peptide 546
antibody. Anti-MERS-CoV nsp1 antibody was generated by immunizing rabbits with purified C-547
terminal His-tagged MERS-CoV nsp1. Vero and 293/CD26 cells were infected with MERS-548
CoV-WT, MERS-CoV-CD, or MERS-CoV-mt at MOI of 3. At indicated time points, whole cell 549
lysates were prepared, and subjected to Western blot analysis as described previously (43). Anti-550
myc antibody (Millipore), anti-tubulin antibody (CALBIOCHEM), or antibodies against each of 551
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
25
MERS-CoV protein described above were used as primary antibodies. Goat anti-mouse 552
immunoglobulin G–horseradish peroxidase or goat anti-rabbit immunoglobulin G–horseradish 553
peroxidase (Santa Cruz) were used as secondary antibodies. 554
555
Total RNA extraction and qRT-PCR Total cellular RNAs were extracted from virus-infected 556
cells by using TRIzol LS reagent (Invitrogen) and Direct-zol RNA MiniPrep (Zymo Research), 557
following instruction manuals. cDNAs were synthesized using SuperScript III reverse 558
transcriptase (Invitrogen) and random primers (Invitrogen). To specifically detect MERS-CoV 559
genomic or subgenomic RNAs, cDNAs were synthesized by using MERS-CoV gene specific 560
primers, 5′-TTTTTTTTCTAATCAGTGTTAACATCAATCATTGG-3′. qRT-PCR was 561
performed using a Bio-Rad CFX96 real-time PCR apparatus and SYBR green Master mix (Bio-562
Rad). PCR conditions were as follows: preincubation at 95°C for 30 s and amplification with 40 563
cycles of 95°C for 15 s and 60°C for 20 s. The purity of the amplified PCR products was 564
confirmed by the dissociation melting curves obtained after each reaction. The primers used for 565
human IFN-β mRNA were 5′-AAGGCCAAGGAGTACAGTC-3′ (forward) and 5′-566
ATCTTCAGTTTVGGAGGTAA-3′ (reverse); the primers for IFN-λ mRNA were 5′-567
CGCCTTGGAAGAGTCACTCA-3′ (forward) and 5′-GAAGCCTCAGGTCCCAATTC-3′ 568
(reverse); the primers for 18S rRNA were 5′-CCGGTACAGTGAAACTGCGAATG-3′ 569
(forward) and 5′-GTTATCCAAGTAGGAGAGGAGCGAG-3′ (reverse); the primers for MERS-570
CoV genomic RNA/mRNA 1 were 5′-AATACACGGTTTCGTCCGGTG-3′ (forward) and 5′-571
ACCACAGAGTGGCACAGTTAG-3′ (reverse); the primers for MERS-CoV subgenomic RNA 572
8 were 5′-CTCGTTCTCTTGCAGAACTTTG-3′ (forward) and 5′-TGCCCAGGTGGAAAGGT-573
3′ (reverse). The relative expression level of each gene mRNA were normalized to 18S rRNA 574
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
26
levels. All of the assays were performed in triplicate, and the results are expressed as means ± 575
the standard deviations. 576
577
Titration of intracellular infectious particles in infected 293/CD26 cells At indicated times 578
p.i., infected 293/CD26 cells were washed two times in PBS and pelleted by centrifugation at 579
3,000 rpm for 10 min. The pelleted cells were suspended in growth medium, and lysed by three 580
freeze-and-thaw cycles. After centrifugation at 3,000 rpm for 10 min, supernatant was collected 581
and subjected to plaque assays using Vero cells. 582
583
Purification of released virus particles After centrifugation at 1,500×g for 10 min at 4°C, 584
supernatants from 293/CD26 cells infected with MERS-CoV-WT, -CD or –mt were irradiated 585
with 2×106 rads from a Gammacell
60Co source (model 109A; J. L. Shepherd and Associates, 586
San Fernando, CA) to completely inactivate viruses. Inactivation of virus infectivity was 587
confirmed by blind passages on the samples in Vero cells two times. The inactivated samples 588
were applied onto a discontinuous sucrose gradient consisted of 20, 30, 50, and 60% sucrose in 589
NTE buffer (100 mM NaCl, 10 mM Tris-HCl [pH 7.5], 1 mM EDTA) and subjected to 590
centrifugation at 26,000 rpm for 3 h in an SW28 rotor. The virus particles in the interface of 50-591
30% fraction were collected, diluted with NTE buffer, applied onto a discontinuous sucrose 592
gradient and centrifuged at 26,000 rpm for 18 h in an SW28 rotor. After collecting the purified 593
MERS-CoV particle in the interface of 50-30% sucrose, MERS-CoV particles were pelleted by 594
centrifugation at 38,000 rpm for 2 h using a Beckman SW41 rotor. The purified virus particles in 595
the pellets were dissolved in the same amount of 1×SDS-sample buffer and subjected to Western 596
blot analysis. 597
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
27
598
Electron microscopic analysis Monolayer 293/CD26 cells were infected with MERS-599
CoV-WT, -CD or -mt at an MOI of 3. At 36 h p.i., cells were washed with PBS and fixed with 600
4% formaldehyde and 0.1% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.3), to which 601
0.03% picric acid and 0.03% CaCl2 were added. The monolayers were washed in 0.1 M 602
cacodylate buffer, and the cells were scraped off and processed further as a pellet. The pellets 603
were postfixed in 1% OsO4 in 0.1 M cacodylate buffer (pH 7.3) for 1 h, washed with distilled 604
water, and en bloc stained with 2% aqueous uranyl acetate for 20 min at 60°C. The pellets were 605
dehydrated in ethanol, processed through propylene oxide, and embedded in Poly/Bed 812 606
(Polysciences). Ultrathin sections were cut on Leica EM UC7 ultramicrotome (Leica 607
Microsystems, Buffalo Grove, IL), stained with lead citrate, and examined with a CM-100 608
electron microscope at 60 kV. 609
610
Statistical Analysis One-way analysis of variance (ANOVA) with Tukey’s multiple-611
comparison test was conducted to determine statistical significance. P value of <0.05 were 612
considered statistically significant. 613
614
Acknowledgement 615
We thank Boyd Yount, Rachel Graham, and Amy Sims in the Baric lab for technical advice 616
regarding reverse genetics system of MERS-CoV. We also thank Julie Wen and Zhixia Ding for 617
their suggestions in electron microscopic analysis. This study was supported by Public Health 618
Service grants AI99107 and AI114657 to SM and AI108197 and AI110700 to RSB from the 619
National Institutes of Health, and a grant from the Institute for Human Infections and Immunity 620
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
28
at The University of Texas Medical Branch to SM. K. Nakagawa was supported by the James W. 621
McLaughlin fellowship fund. 622
623
624
References 625
1. Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. 2012. Isolation of a novel 626 coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 367:1814-20. 627
2. Hui DS, Azhar EI, Kim YJ, Memish ZA, Oh MD, Zumla A. 2018. Middle East respiratory syndrome 628 coronavirus: risk factors and determinants of primary, household, and nosocomial transmission. 629 Lancet Infect Dis doi:10.1016/S1473-3099(18)30127-0. 630
3. Lee HJ, Shieh CK, Gorbalenya AE, Koonin EV, La Monica N, Tuler J, Bagdzhadzhyan A, Lai MM. 631 1991. The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the 632 putative proteases and RNA polymerase. Virology 180:567-82. 633
4. Lomniczi B. 1977. Biological properties of avian coronavirus RNA. J Gen Virol 36:531-3. 634 5. Lomniczi B, Kennedy I. 1977. Genome of infectious bronchitis virus. J Virol 24:99-107. 635 6. Raj VS, Mou H, Smits SL, Dekkers DH, Muller MA, Dijkman R, Muth D, Demmers JA, Zaki A, 636
Fouchier RA, Thiel V, Drosten C, Rottier PJ, Osterhaus AD, Bosch BJ, Haagmans BL. 2013. 637 Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. 638 Nature 495:251-4. 639
7. Burkard C, Verheije MH, Wicht O, van Kasteren SI, van Kuppeveld FJ, Haagmans BL, Pelkmans L, 640 Rottier PJ, Bosch BJ, de Haan CA. 2014. Coronavirus cell entry occurs through the endo-641 /lysosomal pathway in a proteolysis-dependent manner. PLoS Pathog 10:e1004502. 642
8. Snijder EJ, Decroly E, Ziebuhr J. 2016. The Nonstructural Proteins Directing Coronavirus RNA 643 Synthesis and Processing. Adv Virus Res 96:59-126. 644
9. Hurst-Hess KR, Kuo L, Masters PS. 2015. Dissection of amino-terminal functional domains of 645 murine coronavirus nonstructural protein 3. J Virol 89:6033-47. 646
10. Graham RL, Sims AC, Brockway SM, Baric RS, Denison MR. 2005. The nsp2 replicase proteins of 647 murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for 648 viral replication. J Virol 79:13399-411. 649
11. Neuman BW, Chamberlain P, Bowden F, Joseph J. 2014. Atlas of coronavirus replicase structure. 650 Virus Res 194:49-66. 651
12. Masters PS. 2006. The molecular biology of coronaviruses. Adv Virus Res 66:193-292. 652 13. Sawicki SG, Sawicki DL, Siddell SG. 2007. A contemporary view of coronavirus transcription. J 653
Virol 81:20-9. 654 14. Lai MM, Baric RS, Brayton PR, Stohlman SA. 1984. Characterization of leader RNA sequences on 655
the virion and mRNAs of mouse hepatitis virus, a cytoplasmic RNA virus. Proc Natl Acad Sci U S A 656 81:3626-30. 657
15. Lai MM, Patton CD, Baric RS, Stohlman SA. 1983. Presence of leader sequences in the mRNA of 658 mouse hepatitis virus. J Virol 46:1027-33. 659
16. Lai MM, Patton CD, Stohlman SA. 1982. Replication of mouse hepatitis virus: negative-stranded 660 RNA and replicative form RNA are of genome length. J Virol 44:487-92. 661
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
29
17. Liu DX, Fung TS, Chong KK, Shukla A, Hilgenfeld R. 2014. Accessory proteins of SARS-CoV and 662 other coronaviruses. Antiviral Res 109:97-109. 663
18. Narayanan K, Huang C, Makino S. 2008. SARS coronavirus accessory proteins. Virus Res 133:113-664 21. 665
19. Menachery VD, Mitchell HD, Cockrell AS, Gralinski LE, Yount BL, Jr., Graham RL, McAnarney ET, 666 Douglas MG, Scobey T, Beall A, Dinnon K, 3rd, Kocher JF, Hale AE, Stratton KG, Waters KM, Baric 667 RS. 2017. MERS-CoV Accessory ORFs Play Key Role for Infection and Pathogenesis. MBio 8. 668
20. Stertz S, Reichelt M, Spiegel M, Kuri T, Martinez-Sobrido L, Garcia-Sastre A, Weber F, Kochs G. 669 2007. The intracellular sites of early replication and budding of SARS-coronavirus. Virology 670 361:304-15. 671
21. Tooze J, Tooze S, Warren G. 1984. Replication of coronavirus MHV-A59 in sac- cells: 672 determination of the first site of budding of progeny virions. Eur J Cell Biol 33:281-93. 673
22. Klumperman J, Locker JK, Meijer A, Horzinek MC, Geuze HJ, Rottier PJ. 1994. Coronavirus M 674 proteins accumulate in the Golgi complex beyond the site of virion budding. J Virol 68:6523-34. 675
23. Vennema H, Godeke GJ, Rossen JW, Voorhout WF, Horzinek MC, Opstelten DJ, Rottier PJ. 1996. 676 Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral 677 envelope protein genes. EMBO J 15:2020-8. 678
24. de Haan CA, Smeets M, Vernooij F, Vennema H, Rottier PJ. 1999. Mapping of the coronavirus 679 membrane protein domains involved in interaction with the spike protein. J Virol 73:7441-52. 680
25. Escors D, Ortego J, Laude H, Enjuanes L. 2001. The membrane M protein carboxy terminus binds 681 to transmissible gastroenteritis coronavirus core and contributes to core stability. J Virol 682 75:1312-24. 683
26. Escors D, Camafeita E, Ortego J, Laude H, Enjuanes L. 2001. Organization of two transmissible 684 gastroenteritis coronavirus membrane protein topologies within the virion and core. J Virol 685 75:12228-40. 686
27. Kuo L, Masters PS. 2002. Genetic evidence for a structural interaction between the carboxy 687 termini of the membrane and nucleocapsid proteins of mouse hepatitis virus. J Virol 76:4987-99. 688
28. Nguyen VP, Hogue BG. 1997. Protein interactions during coronavirus assembly. J Virol 71:9278-689 84. 690
29. Opstelten DJ, Raamsman MJ, Wolfs K, Horzinek MC, Rottier PJ. 1995. Envelope glycoprotein 691 interactions in coronavirus assembly. J Cell Biol 131:339-49. 692
30. Corse E, Machamer CE. 2000. Infectious bronchitis virus E protein is targeted to the Golgi 693 complex and directs release of virus-like particles. J Virol 74:4319-26. 694
31. Bos EC, Luytjes W, van der Meulen HV, Koerten HK, Spaan WJ. 1996. The production of 695 recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus. 696 Virology 218:52-60. 697
32. Fischer F, Stegen CF, Masters PS, Samsonoff WA. 1998. Analysis of constructed E gene mutants 698 of mouse hepatitis virus confirms a pivotal role for E protein in coronavirus assembly. J Virol 699 72:7885-94. 700
33. DeDiego ML, Alvarez E, Almazan F, Rejas MT, Lamirande E, Roberts A, Shieh WJ, Zaki SR, 701 Subbarao K, Enjuanes L. 2007. A severe acute respiratory syndrome coronavirus that lacks the E 702 gene is attenuated in vitro and in vivo. J Virol 81:1701-13. 703
34. Narayanan K, Ramirez SI, Lokugamage KG, Makino S. 2015. Coronavirus nonstructural protein 1: 704 Common and distinct functions in the regulation of host and viral gene expression. Virus Res 705 202:89-100. 706
35. Connor RF, Roper RL. 2007. Unique SARS-CoV protein nsp1: bioinformatics, biochemistry and 707 potential effects on virulence. Trends Microbiol 15:51-3. 708
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
30
36. Jansson AM. 2013. Structure of alphacoronavirus transmissible gastroenteritis virus nsp1 has 709 implications for coronavirus nsp1 function and evolution. J Virol 87:2949-55. 710
37. Snijder EJ, Bredenbeek PJ, Dobbe JC, Thiel V, Ziebuhr J, Poon LL, Guan Y, Rozanov M, Spaan WJ, 711 Gorbalenya AE. 2003. Unique and conserved features of genome and proteome of SARS-712 coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Biol 331:991-1004. 713
38. Thiel V, Ivanov KA, Putics A, Hertzig T, Schelle B, Bayer S, Weissbrich B, Snijder EJ, Rabenau H, 714 Doerr HW, Gorbalenya AE, Ziebuhr J. 2003. Mechanisms and enzymes involved in SARS 715 coronavirus genome expression. J Gen Virol 84:2305-15. 716
39. Tohya Y, Narayanan K, Kamitani W, Huang C, Lokugamage K, Makino S. 2009. Suppression of 717 host gene expression by nsp1 proteins of group 2 bat coronaviruses. J Virol 83:5282-8. 718
40. Kamitani W, Huang C, Narayanan K, Lokugamage KG, Makino S. 2009. A two-pronged strategy to 719 suppress host protein synthesis by SARS coronavirus Nsp1 protein. Nat Struct Mol Biol 16:1134-720 40. 721
41. Kamitani W, Narayanan K, Huang C, Lokugamage K, Ikegami T, Ito N, Kubo H, Makino S. 2006. 722 Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression 723 by promoting host mRNA degradation. Proc Natl Acad Sci U S A 103:12885-90. 724
42. Huang C, Lokugamage KG, Rozovics JM, Narayanan K, Semler BL, Makino S. 2011. 725 Alphacoronavirus transmissible gastroenteritis virus nsp1 protein suppresses protein translation 726 in mammalian cells and in cell-free HeLa cell extracts but not in rabbit reticulocyte lysate. J Virol 727 85:638-43. 728
43. Lokugamage KG, Narayanan K, Nakagawa K, Terasaki K, Ramirez SI, Tseng CT, Makino S. 2015. 729 Middle East Respiratory Syndrome Coronavirus nsp1 Inhibits Host Gene Expression by 730 Selectively Targeting mRNAs Transcribed in the Nucleus while Sparing mRNAs of Cytoplasmic 731 Origin. J Virol 89:10970-81. 732
44. Gaglia MM, Covarrubias S, Wong W, Glaunsinger BA. 2012. A common strategy for host RNA 733 degradation by divergent viruses. J Virol 86:9527-30. 734
45. Huang C, Lokugamage KG, Rozovics JM, Narayanan K, Semler BL, Makino S. 2011. SARS 735 coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mRNAs: viral 736 mRNAs are resistant to nsp1-induced RNA cleavage. PLoS Pathog 7:e1002433. 737
46. Narayanan K, Huang C, Lokugamage K, Kamitani W, Ikegami T, Tseng CT, Makino S. 2008. Severe 738 acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of 739 type I interferon, in infected cells. J Virol 82:4471-9. 740
47. Wathelet MG, Orr M, Frieman MB, Baric RS. 2007. Severe acute respiratory syndrome 741 coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain. J 742 Virol 81:11620-33. 743
48. Zhang Q, Ke H, Blikslager A, Fujita T, Yoo D. 2018. Type III Interferon Restriction by Porcine 744 Epidemic Diarrhea Virus and the Role of Viral Protein nsp1 in IRF1 Signaling. J Virol 92. 745
49. Zust R, Cervantes-Barragan L, Kuri T, Blakqori G, Weber F, Ludewig B, Thiel V. 2007. Coronavirus 746 non-structural protein 1 is a major pathogenicity factor: implications for the rational design of 747 coronavirus vaccines. PLoS Pathog 3:e109. 748
50. Zhang R, Li Y, Cowley TJ, Steinbrenner AD, Phillips JM, Yount BL, Baric RS, Weiss SR. 2015. The 749 nsp1, nsp13, and M proteins contribute to the hepatotropism of murine coronavirus JHM.WU. J 750 Virol 89:3598-609. 751
51. Jimenez-Guardeno JM, Regla-Nava JA, Nieto-Torres JL, DeDiego ML, Castano-Rodriguez C, 752 Fernandez-Delgado R, Perlman S, Enjuanes L. 2015. Identification of the Mechanisms Causing 753 Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine. 754 PLoS Pathog 11:e1005215. 755
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
31
52. Tanaka T, Kamitani W, DeDiego ML, Enjuanes L, Matsuura Y. 2012. Severe acute respiratory 756 syndrome coronavirus nsp1 facilitates efficient propagation in cells through a specific 757 translational shutoff of host mRNA. J Virol 86:11128-37. 758
53. Terada Y, Kawachi K, Matsuura Y, Kamitani W. 2017. MERS coronavirus nsp1 participates in an 759 efficient propagation through a specific interaction with viral RNA. Virology 511:95-105. 760
54. Scobey T, Yount BL, Sims AC, Donaldson EF, Agnihothram SS, Menachery VD, Graham RL, 761 Swanstrom J, Bove PF, Kim JD, Grego S, Randell SH, Baric RS. 2013. Reverse genetics with a full-762 length infectious cDNA of the Middle East respiratory syndrome coronavirus. Proc Natl Acad Sci 763 U S A 110:16157-62. 764
55. Tseng CT, Tseng J, Perrone L, Worthy M, Popov V, Peters CJ. 2005. Apical entry and release of 765 severe acute respiratory syndrome-associated coronavirus in polarized Calu-3 lung epithelial 766 cells. J Virol 79:9470-9. 767
56. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. 1982. Growth of human hepatoma cells 768 lines with differentiated functions in chemically defined medium. Cancer Res 42:3858-63. 769
57. Mou H, Raj VS, van Kuppeveld FJ, Rottier PJ, Haagmans BL, Bosch BJ. 2013. The receptor binding 770 domain of the new Middle East respiratory syndrome coronavirus maps to a 231-residue region 771 in the spike protein that efficiently elicits neutralizing antibodies. J Virol 87:9379-83. 772
58. Hume AJ, Ames J, Rennick LJ, Duprex WP, Marzi A, Tonkiss J, Muhlberger E. 2016. Inactivation of 773 RNA Viruses by Gamma Irradiation: A Study on Mitigating Factors. Viruses 8. 774
59. Lomax ME, Folkes LK, O'Neill P. 2013. Biological consequences of radiation-induced DNA 775 damage: relevance to radiotherapy. Clin Oncol (R Coll Radiol) 25:578-85. 776
60. Ohshima H, Iida Y, Matsuda A, Kuwabara M. 1996. Damage induced by hydroxyl radicals 777 generated in the hydration layer of gamma-irradiated frozen aqueous solution of DNA. J Radiat 778 Res 37:199-207. 779
61. Summers WC, Szybalski W. 1967. Gamma-irradiation of deoxyribonucleic acid in dilute solutions. 780 II. Molecular mechanisms responsible for inactivation of phage, its transfecting DNA, and of 781 bacterial transforming activity. J Mol Biol 26:227-35. 782
62. Ward RL. 1980. Mechanisms of poliovirus inactivation by the direct and indirect effects of 783 ionizing radiation. Radiat Res 83:330-44. 784
63. Ruch TR, Machamer CE. 2012. The coronavirus E protein: assembly and beyond. Viruses 4:363-785 82. 786
64. Baudoux P, Carrat C, Besnardeau L, Charley B, Laude H. 1998. Coronavirus pseudoparticles 787 formed with recombinant M and E proteins induce alpha interferon synthesis by leukocytes. J 788 Virol 72:8636-43. 789
65. Wang SM, Huang KJ, Wang CT. 2014. BST2/CD317 counteracts human coronavirus 229E 790 productive infection by tethering virions at the cell surface. Virology 449:287-96. 791
66. Neil SJ, Zang T, Bieniasz PD. 2008. Tetherin inhibits retrovirus release and is antagonized by HIV-792 1 Vpu. Nature 451:425-30. 793
67. Kaletsky RL, Francica JR, Agrawal-Gamse C, Bates P. 2009. Tetherin-mediated restriction of 794 filovirus budding is antagonized by the Ebola glycoprotein. Proc Natl Acad Sci U S A 106:2886-91. 795
68. Mansouri M, Viswanathan K, Douglas JL, Hines J, Gustin J, Moses AV, Fruh K. 2009. Molecular 796 mechanism of BST2/tetherin downregulation by K5/MIR2 of Kaposi's sarcoma-associated 797 herpesvirus. J Virol 83:9672-81. 798
69. Jouvenet N, Neil SJ, Zhadina M, Zang T, Kratovac Z, Lee Y, McNatt M, Hatziioannou T, Bieniasz PD. 799 2009. Broad-spectrum inhibition of retroviral and filoviral particle release by tetherin. J Virol 800 83:1837-44. 801
70. Hayashi T, MacDonald LA, Takimoto T. 2015. Influenza A Virus Protein PA-X Contributes to Viral 802 Growth and Suppression of the Host Antiviral and Immune Responses. J Virol 89:6442-52. 803
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
32
71. Xu G, Zhang X, Liu Q, Bing G, Hu Z, Sun H, Xiong X, Jiang M, He Q, Wang Y, Pu J, Guo X, Yang H, 804 Liu J, Sun Y. 2017. PA-X protein contributes to virulence of triple-reassortant H1N2 influenza 805 virus by suppressing early immune responses in swine. Virology 508:45-53. 806
72. Lee J, Yu H, Li Y, Ma J, Lang Y, Duff M, Henningson J, Liu Q, Li Y, Nagy A, Bawa B, Li Z, Tong G, 807 Richt JA, Ma W. 2017. Impacts of different expressions of PA-X protein on 2009 pandemic H1N1 808 virus replication, pathogenicity and host immune responses. Virology 504:25-35. 809
73. Tigges MA, Leng S, Johnson DC, Burke RL. 1996. Human herpes simplex virus (HSV)-specific CD8+ 810 CTL clones recognize HSV-2-infected fibroblasts after treatment with IFN-gamma or when virion 811 host shutoff functions are disabled. J Immunol 156:3901-10. 812
74. Pasieka TJ, Lu B, Crosby SD, Wylie KM, Morrison LA, Alexander DE, Menachery VD, Leib DA. 2008. 813 Herpes simplex virus virion host shutoff attenuates establishment of the antiviral state. J Virol 814 82:5527-35. 815
75. Murphy JA, Duerst RJ, Smith TJ, Morrison LA. 2003. Herpes simplex virus type 2 virion host 816 shutoff protein regulates alpha/beta interferon but not adaptive immune responses during 817 primary infection in vivo. J Virol 77:9337-45. 818
76. Finnen RL, Zhu M, Li J, Romo D, Banfield BW. 2016. Herpes Simplex Virus 2 Virion Host Shutoff 819 Endoribonuclease Activity Is Required To Disrupt Stress Granule Formation. J Virol 90:7943-55. 820
77. Dauber B, Pelletier J, Smiley JR. 2011. The herpes simplex virus 1 vhs protein enhances 821 translation of viral true late mRNAs and virus production in a cell type-dependent manner. J 822 Virol 85:5363-73. 823
78. Finnen RL, Hay TJ, Dauber B, Smiley JR, Banfield BW. 2014. The herpes simplex virus 2 virion-824 associated ribonuclease vhs interferes with stress granule formation. J Virol 88:12727-39. 825
79. Dauber B, Poon D, Dos Santos T, Duguay BA, Mehta N, Saffran HA, Smiley JR. 2016. The Herpes 826 Simplex Virus Virion Host Shutoff Protein Enhances Translation of Viral True Late mRNAs 827 Independently of Suppressing Protein Kinase R and Stress Granule Formation. J Virol 90:6049-57. 828
80. Agrawal AS, Garron T, Tao X, Peng BH, Wakamiya M, Chan TS, Couch RB, Tseng CT. 2015. 829 Generation of a transgenic mouse model of Middle East respiratory syndrome coronavirus 830 infection and disease. J Virol 89:3659-70. 831
81. Falzarano D, de Wit E, Rasmussen AL, Feldmann F, Okumura A, Scott DP, Brining D, Bushmaker T, 832 Martellaro C, Baseler L, Benecke AG, Katze MG, Munster VJ, Feldmann H. 2013. Treatment with 833 interferon-alpha2b and ribavirin improves outcome in MERS-CoV-infected rhesus macaques. Nat 834 Med 19:1313-7. 835
82. Falzarano D, de Wit E, Feldmann F, Rasmussen AL, Okumura A, Peng X, Thomas MJ, van 836 Doremalen N, Haddock E, Nagy L, LaCasse R, Liu T, Zhu J, McLellan JS, Scott DP, Katze MG, 837 Feldmann H, Munster VJ. 2014. Infection with MERS-CoV causes lethal pneumonia in the 838 common marmoset. PLoS Pathog 10:e1004250. 839
83. Cockrell AS, Yount BL, Scobey T, Jensen K, Douglas M, Beall A, Tang XC, Marasco WA, Heise MT, 840 Baric RS. 2016. A mouse model for MERS coronavirus-induced acute respiratory distress 841 syndrome. Nat Microbiol 2:16226. 842
84. Abernathy E, Glaunsinger B. 2015. Emerging roles for RNA degradation in viral replication and 843 antiviral defense. Virology 479-480:600-8. 844
85. Read GS. 2013. Virus-encoded endonucleases: expected and novel functions. Wiley Interdiscip 845 Rev RNA 4:693-708. 846
847
848
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
33
849
Figure Legends 850
Fig. 1. Characterization of loss-of-function mutant, MERS-CoV nsp1-mt, in expressed cells. 851
(A) 293 cells were transfected with 2 g of capped and polyadenylated RNA transcripts 852
encoding CAT, SARS-CoV nsp1, MERS-CoV nsp1-WT, MERS-CoV nsp1-CD, or MERS-CoV 853
nsp1-mt, all of which carried a C-terminal myc epitope tag, radiolabeled with Tran35
S-label from 854
8.5 to 9.5 h post-transfection. Lysates were resolved on 12% SDS-PAGE, followed by 855
autoradiography (top panel), colloidal Coomassie blue staining (middle panel), and Western blot 856
analysis using anti-myc and tubulin antibodies (bottom two panels). (B) 293 cells were 857
transfected with RNA transcripts as described in (A). Intracellular RNAs were extracted at 9 h 858
post-transfection and subjected to Northern blot analysis using a probe for GAPDH mRNA (top). 859
The 28S and 18S rRNAs were detected by ethidium bromide staining (bottom). (C) A schematic 860
diagram of Ren-EMCV-FF is shown at the top of the panel. 293 cells were cotransfected with a 861
plasmid encoding Ren-EMCV-FF and the plasmid expressing CAT, SARS-CoV nsp1, MERS-862
CoV nsp1-WT, MERS-CoV nsp1-CD, or MERS-CoV nsp1-mt protein; all nsp1s carried the C-863
terminal myc tag. At 24 h post-transfection, intracellular RNAs were extracted and subjected to 864
Northern blot analysis using an RNA probe that binds to the rLuc gene (second panel). 865
Arrowhead, full-length Ren-EMCV-FF; arrow, cleaved RNA fragment. The 28S and 18S rRNAs 866
were detected by ethidium bromide staining (third panel). Cell extracts, prepared at 24 h post-867
transfection, were used for Western blot analysis, using anti-myc and tubulin antibodies (fourth 868
and fifth panels). 869
870
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
34
Fig. 2. Growth kinetics of MERS-CoV-WT, -CD, and –mt and accumulation of viral 871
proteins and RNA in infected Vero cells. (A) Vero cells were infected with MERS-CoV-WT 872
(WT), MERS-CoV-CD (CD), or MERS-CoV-mt (mt) at an MOI of 0.01 (left panel) or 3 (right 873
panel). Culture supernatants were collected at the indicated times, and virus titers were 874
determined by plaque assay in Vero cells. The results represent the averages of three independent 875
experiments. Each bar represents the mean (±standard deviation) for three samples. (B) Vero 876
cells were infected with each of the three viruses at an MOI of 3. At indicated times p.i., total 877
proteins were extracted and Western blot analysis was performed to detect the S, M, N, nsp1, and 878
tubulin by using anti-MERS-CoV S, M, N, nsp1, and tubulin antibody, respectively. (C) Vero 879
cells were infected with each of the three viruses at an MOI of 3. At the indicated times, total 880
RNAs were extracted. The viral mRNAs were detected by Northern blot analysis using a probe 881
that binds to the 3’-end of the MERS-CoV genome. The 28S and 18S rRNAs were detected by 882
ethidium bromide staining. 883
884
Fig. 3. Effect of replication of MERS-CoV-WT, MERS-CoV-CD, and MERS-CoV--mt on 885
abundance of host endogenous mRNA and host protein synthesis in Vero cells. (A, B) Vero 886
cells were either mock infected (Mock) or infected with MERS-CoV-WT (WT), MERS-CoV-887
CD (CD), or MERS-CoV-mt (mt) at an MOI of 3. At 16 h (A) or 24 h p.i. (B, left panel), 888
intracellular RNAs were extracted. For testing host mRNA decay in infected cells, ActD was 889
added to the culture at 1 h p.i., and intracellular RNAs were extracted at 24 h p.i. (B, right panel). 890
The abundance of GAPDH mRNA was determined using Northern blot analysis (top panel). The 891
28S and 18S rRNAs were detected by ethidium bromide staining (bottom panel). (C) Vero cells 892
were either mock infected (Mock) or infected with MERS-CoV-WT (WT), MERS-CoV-CD 893
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
35
(CD), or MERS-CoV-mt (mt) at an MOI of 3. The cells were radiolabeled for 1 h with Tran35
S-894
label, and cell lysates were prepared at the indicated times p.i. Cell lysates were subjected to 895
SDS-PAGE analysis, followed by autoradiography (top panel) and colloidal Coomassie blue 896
staining (bottom panel). 897
898
Fig. 4. Replication kinetics of MERS-CoV-WT, MERS-CoV-CD, and MERS-CoV-mt in 899
various cell lines. Calu-3 cells (A), Huh7-cells (B), 293/CD26 cells (C), and HeLa/CD26 cells 900
(D) were infected with MERS-CoV-WT (WT), MERS-CoV-CD (CD), or MERS-CoV-mt (mt) at 901
an MOI of 0.01 (left panels) or MOI of 3 (right panels). Culture supernatants were collected at 902
the indicated times, and virus titers were determined by plaque assay in Vero cells. The results 903
represent the averages of three independent experiments. Each bar represents the mean 904
(±standard deviation) for three samples. Asterisks represent statically significant differences 905
between the titers of MERS-CoV-WT and mutant viruses (P<0.05). 906
907
Fig. 5. IFN-β and IFN –λ mRNA expression in infected 293/CD26 cells. 293/CD26 cells 908
were either mock infected (M) or infected with MERS-CoV-WT (WT), MERS-CoV-CD (CD), 909
or MERS-CoV-mt at an MOI of 3. SeV infection (100 HA units) was inoculated as a positive 910
control. Total intracellular RNAs were extracted at the indicated times, and the amounts of 911
endogenous IFN-β and -λ mRNA mRNAs were determined by qRT-PCR analysis. Expression 912
levels of the genes were normalized to levels of 18S rRNA. Each bar represents the mean 913
(±standard deviation) for three wells. 914
915
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
36
Fig. 6. Accumulation of viral proteins and RNA in infected 293/CD26 cells. (A) 293/CD26 916
cells were either mock infected (Mock) or infected with MERS-CoV-WT (WT), MERS-CoV-917
CD (CD), or MERS-CoV-mt (mt) at an MOI of 3. At indicated times p.i., total proteins were 918
extracted (A) or total RNAs were extracted (B, C). (A) Western blot analysis was performed to 919
detect the S, M, N, nsp1, and tubulin. (B) The viral mRNAs were detected by Northern blot 920
analysis using a probe that binds to the 3’-end of the MERS-CoV genome, and the 28S and 18S 921
rRNAs were detected by ethidium bromide staining. (C) Amounts of genomic RNA and 922
subgenomic mRNA 8 were quantified by qRT-PCR and expression levels were normalized to 923
levels of 18s rRNA. Each bar represents the mean (±standard deviation) for three wells. 924
925
Fig. 7. Effects of virus replication on GAPDH mRNA level and host protein synthesis in 926
293/CD26 cells. 293/CD26 cells were either mock-infected (Mock) or infected with MERS-927
CoV-WT (WT), MERS-CoV-CD (CD), or MERS-CoV-mt (mt) at an MOI of 3. (A) The 928
abundance of GAPDH mRNA at 24 h p.i. was determined using Northern blot analysis. The 28S 929
and 18S rRNAs were detected by ethidium bromide staining. (B) Cells were radiolabeled for 1 h 930
with Tran35
S-label, and cell lysates were prepared at the indicated times p.i. Cell lysates were 931
subjected to SDS-PAGE analysis, followed by autoradiography (top panel) and colloidal 932
Coomassie blue staining (bottom panel). 933
934
Fig. 8. MERS-CoV-CD and MERS-CoV-mt undergo inefficient virus assembly in 935
293/CD26 cells. 293/CD26 cells were infected with MERS-CoV-WT (WT), MERS-CoV-CD 936
(CD), or MERS-CoV-mt (mt) at an MOI of 3. (A) At the indicated time points p.i., the titers of 937
cell-associated viruses were determined by plaque assay. The results represent the averages of 938
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
37
three independent experiments. Each bar represents the mean (±standard deviation) for three 939
samples. Asterisks represent statically significant differences between the titers of MERS-CoV-940
WT and mutant viruses (P<0.05). Hash marks represent statically significant differences between 941
the titers of MERS-CoV-CD and MERS-CoV-mt (P<0.05). (B) At the indicated times p.i., total 942
proteins were extracted, and subjected to Western blot analysis using anti-human CD26 or 943
tubulin antibody. Lysate of mock-infected 293 cells was included as a negative control in the far 944
right lane of bottom panels. (C) At 36 h p.i., supernatants were collected and subject to 60
Co-945
irradiation. The purified viruses were pelleted, dissolved in the same volume of sample buffer, 946
and subjected to Western blot analysis by using anti-S, M, and N protein antibodies. 947
948
Fig. 9. Transmission electron microscopy of 293/CD26 cells infected with MERS-CoV-WT, 949
-CD, or –mt. Ultrastructure analyses of 293/CD26 cells infected with MERS-CoV-WT (A), -950
CD (B), or –mt (C). Arrowheads indicate vesicles containing virus particles. Bars, 0.5 µm. (D) 951
Numbers of virus particles in randomly selected 30 vesicles for each virus sample. Each dot 952
represent the number of virus particles in each vesicle. Asterisks represent statically significant 953
differences in virus titers (P<0.05). ns, not significance. 954
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
Nakagawa et al.,
Fig. 1
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
Nakagawa et al.,
Fig. 2
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
Nakagawa et al.,
Fig. 3
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
Nakagawa et al.,
Fig. 4
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
Nakagawa et al.,
Fig. 5
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
Nakagawa et al.,
Fig. 6
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
Nakagawa et al.,
Fig. 7
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
Nakagawa et al.,
Fig. 8
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
Nakagawa et al.,
Fig. 9
on August 17, 2018 by guest
http://jvi.asm.org/
Dow
nloaded from
top related